Calcium signal pathways are used extensively to trigger rapid changes in cellular activity. The extent to which calcium signals are utilized by Trypanosoma brucei to coordinate complex life cycle events, and the accessibility of the signal pathways to pharmaceutical disruption is poorly understood. Progress in this area has been slow due to the absence of a suitable test system to monitor signal transduction across the plasma membrane and release of calcium from intracellular compartments. Only intact cells can be used for these studies. Dr. Ruben's laboratory utilized a systematic approach to solve this problem. Intact trypanosomes were loaded with the calcium sensitive fluorochrome Fura-2 to monitor intracellular free calcium concentrations (Ca2+). This system was used to identify amphipathic peptides and amines that opened a G protein- coupled calcium channel, and released stored calcium. The unique ability to selectively open trypanosome calcium channels was then exploited to identify pharmaceutical agents that disrupted channel activity. Two novel antagonist were discovered. The present proposal builds upon these results. Specific Aim 1, is to characterize the organelles and energy dependent proteins that maintain calcium homeostasis. Sustained disruption of calcium homeostasis is lethal to eukaryotic cells. Isolated organelles will be used to identify which compartments are capable of calcium transport, and the mechanisms of calcium influx and eflux. Emphasis is placed upon the lysosomes, endoplasmic reticulum and mitochondria. Intact cells that over-express the endoplasmic reticulum Ca2+-ATPase will be used to understand the contribution of this organelle to calcium homeostasis in vivo. The calcium-sensitive luminescent protein, aequorin will be fused to a mitochondrial target signal to report on mitochondrial calcium in the intact trypanosome. Specific Aim 2, is to characterize at the molecular level properties of two proteins that modulate calcium channel activity; the G protein, and benzylated triazole receptor. The location of these proteins in the cell, the identification of functional domains, and structural requirement for drug binding will be determined. Specific Aim 3, is to evaluate the pharmacology of the trypanosome calcium channels. Benzylated triazole disrupt the activity of trypanosome calcium channels. In collaboration with an organic chemist, radioactive derivatives of these compounds will be synthesized, along with whose side chains contain different functional groups. The derivatives will be used to quantitate the benzylated triazole receptor number and affinity: as an affinity tag to follow the receptor protein during purification; and to evaluate side chain requirements for drug interactions with the receptor. The effects of these drugs on acute and chronic infections in mice will be determined.